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1.
Cancer Res ; 61(18): 6669-73, 2001 Sep 15.
Article in English | MEDLINE | ID: mdl-11559532

ABSTRACT

Solid tumors contain regions of hypoxia, a physiological stress that can activate cell death pathways and, thus, result in the selection of cells resistant to death signals and anticancer therapy. Bcl2/adenovirus EIB 19kD-interacting protein 3 (BNIP3) is a cell death factor that is a member of the Bcl-2 proapoptotic family recently shown to induce necrosis rather than apoptosis. Using cDNA arrays and serial analysis of gene expression, we found that hypoxia induces up-regulation of BNIP3 and its homologue, Nip3-like protein X. Analysis of human carcinoma cell lines showed that they are hypoxically regulated in many tumor types, as well as in endothelial cells and macrophages. Regulation was hypoxia inducible factor-1-dependent, and hypoxia inducible factor-1 expression was suppressed by von Hippel-Lindau protein in normoxic cells. Northern blotting and in situ hybridization analysis has revealed that these factors are highly expressed in human tumors compared with normal tissue and that BNIP3 is up-regulated in perinecrotic regions of the tumor. This study shows that genes regulating cell death can be hypoxically induced and are overexpressed in clinical tumors.


Subject(s)
DNA-Binding Proteins/physiology , Membrane Proteins/biosynthesis , Nuclear Proteins/physiology , Proto-Oncogene Proteins , Transcription Factors , Tumor Suppressor Proteins , Animals , Apoptosis/physiology , Breast/metabolism , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , CHO Cells , Carcinoma, Renal Cell/genetics , Carcinoma, Renal Cell/metabolism , Cell Hypoxia/physiology , Cricetinae , DNA-Binding Proteins/biosynthesis , Gene Expression Regulation, Neoplastic , Humans , Hypoxia-Inducible Factor 1 , Hypoxia-Inducible Factor 1, alpha Subunit , Kidney Neoplasms/genetics , Kidney Neoplasms/metabolism , Membrane Proteins/genetics , Nuclear Proteins/biosynthesis , Oligonucleotide Array Sequence Analysis , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Tumor Cells, Cultured , Up-Regulation
2.
Proc Natl Acad Sci U S A ; 98(10): 5746-51, 2001 May 08.
Article in English | MEDLINE | ID: mdl-11331782

ABSTRACT

Cytotoxic T lymphocytes (CTL) induce apoptosis by engaging death receptors or by exocytosis of cytolytic granules containing granzyme (Gzm) proteases and perforin. The lamins, which maintain the structural integrity of the nuclear envelope, are cleaved by caspases during caspase-mediated apoptosis. Although death receptor engagement and GzmB activate caspases, CTL also induce apoptosis during caspase blockade. Both GzmA and GzmB directly and efficiently cleave laminB in vitro, in situ in isolated nuclei and in cells loaded with perforin and Gzms, even in the presence of caspase inhibitors. LaminB is cleaved by GzmA at concentrations of 3 nM, but GzmB is 50 times less active. GzmA cuts laminB at R392; GzmB cuts at the caspase VEVD231 site. Characteristic laminB fragments generated by Gzm proteolysis also are observed during CTL lysis, even in the presence of caspase inhibitors or in cells overexpressing bcl-2. Lamins A/C are direct substrates of GzmA, but not GzmB. GzmA and GzmB therefore directly target critical caspase substrates in caspase-resistant cells.


Subject(s)
Cell Nucleus/metabolism , Nuclear Proteins/metabolism , Serine Endopeptidases/metabolism , Cell Line , Cytoplasmic Granules/metabolism , Granzymes , Hydrolysis , Lamins , T-Lymphocytes, Cytotoxic/immunology
3.
Cell Death Differ ; 8(3): 219-24, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11319604

ABSTRACT

Apoptosis and necrosis need to be differentiated in order to distinguish drug-induced cell death from spontaneous cell death due to hypoxia. The ability to differentiate between these two modes of cell death, especially at an early stage in the process, could have a significant impact on accessing the outcome of anticancer drug therapy in the clinic. Nuclear magnetic resonance spectroscopy was used to distinguish apoptosis from necrosis in human cervical carcinoma (HeLa) cells. Apoptosis was induced by treatment with the topoisomerase II inhibitor etoposide, whereas necrosis was induced by the use of ethacrynic acid or cytochalasin B. We found that the intensity of the methylene resonance increases significantly as early as 6 h after the onset of apoptosis, but that no such changes occur during necrosis. The spectral intensity ratio of the methylene to methyl resonances also shows a high correlation with the percentage of apoptotic cells in the sample (r2=0.965, P<0.003).


Subject(s)
Apoptosis/drug effects , Magnetic Resonance Spectroscopy/methods , Uterine Cervical Neoplasms/pathology , Cytochalasin B/pharmacology , Ethacrynic Acid/pharmacology , Female , HeLa Cells , Humans , Necrosis/chemically induced , Protons , Uterine Cervical Neoplasms/drug therapy
4.
J Biol Chem ; 276(10): 6974-82, 2001 Mar 09.
Article in English | MEDLINE | ID: mdl-11114298

ABSTRACT

Many cell death pathways converge at the mitochondria to induce release of apoptogenic proteins and permeability transition, resulting in the activation of effector caspases responsible for the biochemical and morphological alterations of apoptosis. The death receptor pathway has been described as a triphasic process initiated by the activation of apical caspases, a mitochondrial phase, and then the final phase of effector caspase activation. Granzyme B (GrB) activates apical and effector caspases as well as promotes cytochrome c (cyt c) release and loss of mitochondrial membrane potential. We investigated how GrB affects mitochondria utilizing an in vitro cell-free system and determined that cyt c release and permeability transition are initiated by distinct mechanisms. The cleavage of cytosolic BID by GrB results in truncated BID, initiating mitochondrial cyt c release. BID is the sole cytosolic protein responsible for this phenomenon in vitro, yet caspases were found to participate in cyt c release in some cells. On the other hand, GrB acts directly on mitochondria in the absence of cytosolic S100 proteins to open the permeability transition pore and to disrupt the proton electrochemical gradient. We suggest that GrB acts by two distinct mechanisms on mitochondria that ultimately lead to mitochondrial dysfunction and cellular demise.


Subject(s)
Cytochrome c Group/metabolism , Mitochondria/metabolism , Serine Endopeptidases/pharmacology , Animals , Apoptosis , Caspases/metabolism , Cell Line , Cell-Free System , Cytosol/metabolism , Dose-Response Relationship, Drug , Electrochemistry , Enzyme Activation , Fibroblasts/metabolism , Granzymes , HeLa Cells , Humans , Membrane Potentials , Mice , Microscopy, Confocal , Microscopy, Fluorescence , Mutation , Permeability , Rats , Recombinant Proteins/metabolism , S100 Proteins/metabolism , Time Factors
5.
J Biol Chem ; 276(5): 3683-90, 2001 Feb 02.
Article in English | MEDLINE | ID: mdl-11060286

ABSTRACT

The cytotoxic T lymphocyte protease granzyme A induces caspase-independent cell death in which DNA single-strand nicking is observed instead of oligonucleosomal fragmentation. Granzyme A is a specific tryptase that concentrates in the nucleus of targeted cells and synergistically enhances DNA fragmentation induced by the caspase activator granzyme B. Here we show that granzyme A treatment of isolated nuclei enhances DNA accessibility to exogenous endonucleases. In vitro and after cell loading with perforin, GrnA completely degrades histone H1 and cleaves core histones into approximately 16-kDa fragments. Histone digestion provides a mechanism for unfolding compacted chromatin and facilitating endogenous DNase access to DNA during T cell and natural killer cell granule-mediated apoptosis.


Subject(s)
Histones/metabolism , Serine Endopeptidases/metabolism , T-Lymphocytes, Cytotoxic/enzymology , Animals , Bacterial Proteins/metabolism , COS Cells , Caspase Inhibitors , Caspases/metabolism , Cell Nucleus/metabolism , Chromatin/metabolism , DNA/metabolism , DNA-Binding Proteins/metabolism , Deoxyribonucleases/metabolism , Granzymes , HL-60 Cells , Heparin/analogs & derivatives , Heparin/metabolism , Humans , K562 Cells , Membrane Glycoproteins/metabolism , Perforin , Pore Forming Cytotoxic Proteins , Protein Structure, Tertiary/physiology , Proteoglycans/metabolism , Substrate Specificity , T-Lymphocytes, Cytotoxic/metabolism , Trypsin/metabolism
6.
Oncogene ; 19(48): 5453-63, 2000 Nov 16.
Article in English | MEDLINE | ID: mdl-11114722

ABSTRACT

We have studied ceBNIP3, the orthologue of BNIP3 in C. elegans. Sequence analysis reveals that the different domains of BNIP3 have been conserved throughout evolution. ceBNIP3 contains a C-terminal transmembrane (TM) domain, a conserved domain (CD) of 19 amino acids, a BCL-2 homology-3 (BH3)-like domain and a PEST sequence. ceBNIP3 is expressed primarily as a 25 kDa monomer and a 50 kDa homodimer. After transfection, ceBNIP3 protein is rapidly degraded through a ubiquitin-dependent pathway by the proteasome. Like BNIP3, the TM domain of ceBNIP3 mediates the localization of the protein to mitochondria and is also necessary for homodimerization and cell death in mammalian cells. Neither the putative BH3 domain nor conserved domain is necessary for killing. ceBNIP3 protein interacts with CED-9 and BCL-XL, but unlike other pro-apoptotic BCL-2 family members, the BH3-like domain does not participate in dimerization. The ceBNIP3 TM domain mediates interaction with both CED-9 and BCL-XL. ceBNIP3 interacts with CED-3 but co-expression of CED-3 and ceBNIP3 does not significantly enhance induction of cell death in the presence or absence of CED-4. ceBNIP3 kills mammalian cells by a caspase-independent mechanism. In conclusion, we find that although ceBNIP3 interacts with CED-9 and CED-3 it kills by a BH3- and caspase-independent mechanism.


Subject(s)
Apoptosis/physiology , Caenorhabditis elegans Proteins , Carrier Proteins/physiology , Caspases/physiology , Cysteine Endopeptidases/physiology , Helminth Proteins/physiology , Proto-Oncogene Proteins c-bcl-2/physiology , Proto-Oncogene Proteins/physiology , Tumor Suppressor Proteins , Amino Acid Chloromethyl Ketones/chemistry , Amino Acid Sequence , Animals , Apoptosis/drug effects , Apoptosis Regulatory Proteins , Carrier Proteins/genetics , Carrier Proteins/metabolism , Caspase Inhibitors , Cell Line , Conserved Sequence , Cysteine Endopeptidases/metabolism , Cysteine Proteinase Inhibitors/pharmacology , Dimerization , Gene Expression Regulation , Helminth Proteins/metabolism , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Membrane Proteins/physiology , Mice , Molecular Sequence Data , Multienzyme Complexes/physiology , Protein Structure, Tertiary , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Sequence Homology, Amino Acid , Subcellular Fractions/metabolism , bcl-X Protein
7.
Am J Respir Cell Mol Biol ; 23(4): 475-84, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11017912

ABSTRACT

Elevated concentrations of hyaluronan (HA) are associated with the accumulation of macrophages in the lung after injury. We have investigated the role of HA in the inflammatory and fibrotic responses to lung injury using the intratracheal instillation of bleomycin in rats as a model. After bleomycin-induced lung injury, both HA content in bronchoalveolar lavage (BAL) and staining for HA in macrophages accumulating in injured areas of the lung were maximal at 4 d. Increased HA in BAL correlated with increased locomotion of isolated alveolar macrophages. HA-binding peptide was able to specifically block macrophage motility in vitro. Importantly, systemic administration of HA-binding peptide to rats before injury not only decreased alveolar macrophage motility and accumulation in the lung, but also reduced lung collagen alpha (I) messenger RNA and hydroxyproline contents. We propose a model in which HA plays a critical role in the inflammatory response and fibrotic consequences of acute lung injury.


Subject(s)
Bleomycin/adverse effects , Cell Aggregation/physiology , Collagen/metabolism , Hyaluronic Acid/physiology , Lung/drug effects , Macrophages/cytology , Amino Acid Sequence , Animals , Base Sequence , DNA Primers , Hyaluronic Acid/metabolism , Lung/metabolism , Lung/pathology , Male , Peptides/metabolism , Protein Binding , Rats , Rats, Sprague-Dawley
8.
Mol Cell Biol ; 20(15): 5454-68, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10891486

ABSTRACT

Many apoptotic signaling pathways are directed to mitochondria, where they initiate the release of apoptogenic proteins and open the proposed mitochondrial permeability transition (PT) pore that ultimately results in the activation of the caspase proteases responsible for cell disassembly. BNIP3 (formerly NIP3) is a member of the Bcl-2 family that is expressed in mitochondria and induces apoptosis without a functional BH3 domain. We report that endogenous BNIP3 is loosely associated with mitochondrial membrane in normal tissue but fully integrates into the mitochondrial outer membrane with the N terminus in the cytoplasm and the C terminus in the membrane during induction of cell death. Surprisingly, BNIP3-mediated cell death is independent of Apaf-1, caspase activation, cytochrome c release, and nuclear translocation of apoptosis-inducing factor. However, cells transfected with BNIP3 exhibit early plasma membrane permeability, mitochondrial damage, extensive cytoplasmic vacuolation, and mitochondrial autophagy, yielding a morphotype that is typical of necrosis. These changes were accompanied by rapid and profound mitochondrial dysfunction characterized by opening of the mitochondrial PT pore, proton electrochemical gradient (Deltapsim) suppression, and increased reactive oxygen species production. The PT pore inhibitors cyclosporin A and bongkrekic acid blocked mitochondrial dysregulation and cell death. We propose that BNIP3 is a gene that mediates a necrosis-like cell death through PT pore opening and mitochondrial dysfunction.


Subject(s)
Cell Death/genetics , Intracellular Membranes/metabolism , Membrane Proteins/metabolism , Mitochondria/metabolism , Proto-Oncogene Proteins , Tumor Suppressor Proteins , Apoptosis Inducing Factor , Apoptotic Protease-Activating Factor 1 , Bongkrekic Acid/pharmacology , Caspase 3 , Caspase 9 , Caspases/genetics , Caspases/metabolism , Cell Death/drug effects , Cell Line , Cyclosporine/pharmacology , Cytochrome c Group/metabolism , DNA Fragmentation , Fibroblasts/pathology , Fibroblasts/ultrastructure , Flavoproteins/metabolism , HeLa Cells , Humans , Membrane Proteins/drug effects , Membrane Proteins/genetics , Mitochondria/drug effects , Necrosis , Permeability , Proteins/genetics , Proteins/metabolism , Reactive Oxygen Species/metabolism
9.
Methods Enzymol ; 322: 125-43, 2000.
Article in English | MEDLINE | ID: mdl-10914010

ABSTRACT

Granzyme B (GrB) is the primary molecular mediator of apoptosis by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. It is a unique mammalian aspartic acid-cleaving serine protease. On T cell receptor activation, GrB is released from the CTL cytoplasmic granules by exocytosis, enters the target cells and, in the presence of the granule pore-forming protein perforin, it initiates the processing of caspases and apoptosis. GrB apoptosis is also activated by adenovirus, which can effectively replace perforin. Methods for the purification and quantitation of GrB and perforin, and the preparation and titration of adenovirus, are described. In addition, methods for application of these reagents to the initiation of apoptosis in tumor target cells, with several assays for detecting GrB apoptotic activity, are detailed.


Subject(s)
Killer Cells, Natural/enzymology , Serine Endopeptidases/isolation & purification , Serine Endopeptidases/metabolism , T-Lymphocytes, Cytotoxic/enzymology , Animals , Cell Fractionation/methods , Cell Line , Centrifugation, Density Gradient/methods , Chromatography, Affinity , Chromatography, Gel/methods , Chromatography, Liquid , Cytoplasmic Granules/enzymology , Cytoplasmic Granules/ultrastructure , DNA Damage , DNA Fragmentation , Enzyme-Linked Immunosorbent Assay/methods , Granzymes , Hemoglobins/analysis , Hemolysis , Humans , Idoxuridine/analysis , Idoxuridine/pharmacokinetics , Iodine Radioisotopes , Leukemia, Experimental/enzymology , Membrane Glycoproteins/isolation & purification , Membrane Glycoproteins/metabolism , Perforin , Pore Forming Cytotoxic Proteins , Rabbits , Rats , Rats, Inbred F344 , Sheep , Tumor Cells, Cultured
10.
Am J Physiol Regul Integr Comp Physiol ; 278(5): R1321-8, 2000 May.
Article in English | MEDLINE | ID: mdl-10801303

ABSTRACT

The impact of plasma corticosterone levels on the sympathetic nervous system (SNS) response to intravenous lipopolysaccharide (LPS) or intracerebroventricular injections of PG was studied in anesthetized (urethan-chloralose) male Sprague-Dawley rats. For this, electrophysiological recordings of splenic and renal nerves were completed in control or adrenalectomized (ADX) rats. LPS (10 microgram iv) similarly increased splenic and renal nerve activity in control rats with a shorter onset latency for the splenic nerve. Acute ADX enhanced the response of both nerves to LPS (P < 0.005) and reduced the onset latency of the renal nerve (P < 0.05). PGE(2) (2 microgram icv) rapidly increased the activity of both nerves but preferentially (magnitude and onset latency) stimulated the renal nerve (P < 0.05). The magnitude of the splenic nerve response to PGE(2) was unaffected by ADX. Unexpectedly, PGE(2) was less effective at stimulating renal nerve activity in ADX animals relative to intact controls (P < 0.05). Pretreatment of ADX rats with a CRF antagonist ([D-Phe(12), Nle(21,38), Calpha-MeLeu(37)]CRF-(12-41)) reversed this effect such that the renal nerve responded to central PGE(2) to a greater extent than the splenic nerve (P < 0.05), as was the case in non-ADX rats. These data indicate that enhanced sensitivity of central sympathetic pathways does not account for the enhanced SNS responses to LPS in ADX rats. Also, a CRF-related process appears to diminish renal sympathetic outflow in ADX rats.


Subject(s)
Adrenalectomy , Dinoprostone/pharmacology , Lipopolysaccharides/pharmacology , Sympathetic Nervous System/drug effects , Sympathetic Nervous System/physiology , Animals , Blood Pressure/drug effects , Corticosterone/blood , Corticotropin-Releasing Hormone/physiology , Dinoprostone/administration & dosage , Injections, Intravenous , Injections, Intraventricular , Kidney/innervation , Lipopolysaccharides/administration & dosage , Male , Rats , Rats, Sprague-Dawley , Receptors, Corticotropin-Releasing Hormone/antagonists & inhibitors , Receptors, Corticotropin-Releasing Hormone/physiology , Spleen/innervation
11.
J Biol Chem ; 275(2): 1439-48, 2000 Jan 14.
Article in English | MEDLINE | ID: mdl-10625696

ABSTRACT

BNIP3 (formerly NIP3) is a pro-apoptotic, mitochondrial protein classified in the Bcl-2 family based on limited sequence homology to the Bcl-2 homology 3 (BH3) domain and COOH-terminal transmembrane (TM) domain. BNIP3 expressed in yeast and mammalian cells interacts with survival promoting proteins Bcl-2, Bcl-X(L), and CED-9. Typically, the BH3 domain of pro-apoptotic Bcl-2 homologues mediates Bcl-2/Bcl-X(L) heterodimerization and confers pro-apoptotic activity. Deletion mapping of BNIP3 excluded its BH3-like domain and identified the NH(2) terminus (residues 1-49) and TM domain as critical for Bcl-2 heterodimerization, and either region was sufficient for Bcl-X(L) interaction. Additionally, the removal of the BH3-like domain in BNIP3 did not diminish its killing activity. The TM domain of BNIP3 is critical for homodimerization, pro-apoptotic function, and mitochondrial targeting. Several TM domain mutants were found to disrupt SDS-resistant BNIP3 homodimerization but did not interfere with its killing activity or mitochondrial localization. Substitution of the BNIP3 TM domain with that of cytochrome b(5) directed protein expression to nonmitochondrial sites and still promoted apoptosis and heterodimerization with Bcl-2 and Bcl-X(L). We propose that BNIP3 represents a subfamily of Bcl-2-related proteins that functions without a typical BH3 domain to regulate apoptosis from both mitochondrial and nonmitochondrial sites by selective Bcl-2/Bcl-X(L) interactions.


Subject(s)
Apoptosis , Membrane Proteins/metabolism , Mitochondria/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Proto-Oncogene Proteins , Tumor Suppressor Proteins , Amino Acid Sequence , Animals , Antibodies , Dimerization , Fibroblasts , Humans , Kinetics , Membrane Proteins/chemistry , Mitochondria/ultrastructure , Molecular Sequence Data , Protein Multimerization , Proto-Oncogene Proteins c-bcl-2/chemistry , Rats , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , bcl-X Protein , beta-Galactosidase/metabolism
12.
Immunity ; 10(5): 585-94, 1999 May.
Article in English | MEDLINE | ID: mdl-10367904

ABSTRACT

Cytotoxic lymphocytes trigger apoptosis by releasing perforin and granzymes (Grn). GrnB activates the caspase apoptotic pathway, but little is known about GrnA-induced cell death. Perforin was used to load recombinant GrnA and GrnB and enzymatically inactive variants into target cells. GrnA induces single-strand DNA breaks that can be labeled with Klenow polymerase and visualized on alkaline gels. GrnA-induced DNA damage but not cytolysis requires GrnA proteolysis. GrnA-induced membrane perturbation, nuclear condensation, and DNA damage are unimpaired by caspase blockade. GrnA fails to induce cleavage of caspase-3, lamin B, rho-GTPase, or PARP. GrnA-induced cytotoxicity and cleavage of PHAP II, a previously identified GrnA substrate, are unimpaired in Jurkat cells that overexpress bcl-2. Therefore, GrnA activates a novel apoptotic pathway.


Subject(s)
Caspases/metabolism , DNA Damage , Serine Endopeptidases/pharmacology , T-Lymphocytes, Cytotoxic/drug effects , Animals , Apoptosis/drug effects , Cytotoxicity, Immunologic/drug effects , Enzyme Activation , Esterases/metabolism , Gene Expression , Genes, bcl-2/genetics , Granzymes , Humans , K562 Cells/drug effects , Tumor Cells, Cultured
13.
Oncogene ; 18(13): 2181-8, 1999 Apr 01.
Article in English | MEDLINE | ID: mdl-10327064

ABSTRACT

The contact of natural killer (NK) cells with foreign cells and with certain virus-infected or tumor cells triggers the cytolytic machinery of NK cells. This triggering leads to exocytosis of the cytotoxic NK cell granules. The oncoproteins c-Myc and E1A render cells vulnerable to NK cell mediated cytolysis yet the mechanisms of sensitization are not well understood. In a model where foreign cells (rat fibroblasts) were cocultured with human IL-2 activated NK cells, we observed that NK cells were capable of efficiently killing their targets only if the cells overexpressed the oncogene c-Myc or E1A. Both the parental and the oncogene expressing fibroblasts similarly triggered phosphoinositide hydrolysis in the bound NK cells, demonstrating that NK cells were cytolytically activated in contact with both resistant parental and oncogene expressing sensitive target fibroblasts. The cell death was independent of wild-type p53 and was not inhibited by an anti-apoptotic protein EIB19K. These results provided evidence that c-Myc and E1A activated the NK cell induced cytolysis at a post-triggering stage of NK cell-target cell interaction. In consistence, the c-Myc and E1A overexpressing fibroblasts were more sensitive to the cytolytic effects of isolated NK cell-derived granules than parental cells. The data indicate that oncogenes activate the cytotoxicity of NK cell granules. This mechanism can have a role in directing the cytolytic action of NK cells towards the virus-infected and cancer cells.


Subject(s)
Adenovirus E1A Proteins/physiology , Cytoplasmic Granules/metabolism , Cytotoxicity, Immunologic/physiology , Killer Cells, Natural/immunology , Proto-Oncogene Proteins c-myc/physiology , Actins/chemistry , Adenovirus E1A Proteins/genetics , Animals , Apoptosis/genetics , Biopolymers , Cell Adhesion , Cell Membrane/drug effects , Exocytosis , Fas Ligand Protein , Fibroblasts/immunology , Genes, myc , Genes, p53 , Humans , Interleukin-2/pharmacology , Killer Cells, Natural/drug effects , Membrane Glycoproteins/physiology , Phosphatidylinositols/physiology , Rats , Recombinant Fusion Proteins/physiology , Signal Transduction , Transfection , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/physiology , Tumor Suppressor Protein p53/physiology , fas Receptor/physiology
14.
J Exp Med ; 189(1): 131-44, 1999 Jan 04.
Article in English | MEDLINE | ID: mdl-9874570

ABSTRACT

Granzyme B (GraB) is required for the efficient activation of apoptosis by cytotoxic T lymphocytes and natural killer cells. We find that GraB and perforin induce severe mitochondrial perturbation as evidenced by the release of cytochrome c into the cytosol and suppression of transmembrane potential (Deltapsi). The earliest mitochondrial event was the release of cytochrome c, which occurred at the same time as caspase 3 processing and consistently before the activation of apoptosis. Granzyme K/perforin or perforin treatment, both of which kill target cells efficiently but are poor activators of apoptosis in short-term assays, did not induce rapid cytochrome c release. However, they suppressed Deltapsi and increased reactive oxygen species generation, indicating that mitochondrial dysfunction is also associated with this nonapoptotic cell death. Pretreatment with peptide caspase inhibitors zVAD-FMK or YVAD-CHO prevented GraB apoptosis and cytochrome c release, whereas DEVD-CHO blocked apoptosis but did not prevent cytochrome c release, indicating that caspases act both up- and downstream of mitochondria. Of additional interest, Deltapsi suppression mediated by GraK or GraB and perforin was not affected by zVAD-FMK and thus was caspase independent. Overexpression of Bcl-2 and Bcl-XL suppressed caspase activation, mitochondrial cytochrome c release, Deltapsi suppression, and apoptosis and cell death induced by GraB, GraK, or perforin. In an in vitro cell free system, GraB activates nuclear apoptosis in S-100 cytosol at high doses, however the addition of mitochondria amplified GraB activity over 15-fold. GraB- induced caspase 3 processing to p17 in S-100 cytosol was increased only threefold in the presence of mitochondria, suggesting that another caspase(s) participates in the mitochondrial amplification of GraB apoptosis. We conclude that GraB-induced apoptosis is highly amplified by mitochondria in a caspase-dependent manner but that GraB can also initiate caspase 3 processing and apoptosis in the absence of mitochondria.


Subject(s)
Apoptosis/drug effects , Mitochondria/metabolism , Serine Endopeptidases/pharmacology , Animals , Caspases/metabolism , Cell Line , Chymases , Cytochrome c Group/metabolism , Fluorescent Antibody Technique , Genes, bcl-2/genetics , Granzymes , Humans , Membrane Glycoproteins/pharmacology , Membrane Potentials/drug effects , Perforin , Pore Forming Cytotoxic Proteins , Rats , Reactive Oxygen Species/metabolism , Serine Proteinase Inhibitors/pharmacology , Tryptases
15.
Brain Res ; 811(1-2): 111-21, 1998 Nov 16.
Article in English | MEDLINE | ID: mdl-9804916

ABSTRACT

Interleukin (IL)-1, IL-2 and IL-6 influence central monoamine activity in a cytokine-specific manner. We demonstrated that whereas IL-2 increased hypothalamic and hippocampal norepinephrine (NE) utilization, and DA turnover in the prefrontal cortex, IL-6 induced profound elevations of serotonin (5-HT) and mesocortical dopamine (DA) activity in the hippocampus and prefrontal cortex [S. Zalcman, J.M. Green-Johnson, L. Murray, D.M. Nance, D.G. Dyck, H. Anisman, A. H. Greenberg, Cytokine-specific central monoamine alterations following IL-1, -2 and -6 administration, Brain Res. 643 (1994) 40-49]. IL-1, in contrast, induced a wide range of central monoamine alterations. We presently report that these cytokines also differentially influence behavior. Profound reductions in non-ambulatory and ambulatory exploration were induced in BALB/c mice following IL-1 administration. In contrast, IL-2-treated mice displayed significant increases in the time spent engaged in non-ambulatory exploration, digging, rearing (particularly the number of free rears), and in the investigation of a novel stimulus (i.e., increased number and duration of stimulus contacts). IL-6-treated mice, moreover, exhibited significant increases in the time spent engaged in ambulatory exploration, digging and rearing (particularly the number of free rears, which tended to be of short duration). Modest increases in locomotion and grooming were also observed in IL-6-treated animals. Plasma corticosterone levels did not vary significantly as a function of IL-6 treatment. Hence, cytokine-specific behavioral-activating effects were induced following administration of IL-2 and IL-6. We suggest that these effects have adaptive significance and relevance to sickness behavior; however, pathological outcomes (e.g., schizophrenia, anxious-like states, anxious depression, motor abnormalities) could develop should these cytokines be overproduced or dysregulated.


Subject(s)
Behavior, Animal/drug effects , Interleukin-2/pharmacology , Interleukin-6/pharmacology , Adaptation, Physiological , Animals , Corticosterone/blood , Exploratory Behavior/drug effects , Male , Mental Disorders/physiopathology , Mice , Mice, Inbred BALB C , Motor Activity/drug effects
16.
J Biol Chem ; 273(37): 23946-51, 1998 Sep 11.
Article in English | MEDLINE | ID: mdl-9727009

ABSTRACT

Nuclear factor kappaB (NFkappaB) is a ubiquitously expressed transcription factor that is regulated by the cytoplasmic inhibitor protein IkappaBalpha. Biological agents such as tumor necrosis factor alpha (TNFalpha), which activate NFkappaB, result in the rapid degradation of IkappaBalpha. Adenoviral-mediated gene transfer of Bcl-2 prevents apoptosis of neonatal ventricular myocytes induced by TNFalpha. In view of the growing evidence that NFkappaB may play an important role in regulating apoptosis, we determined whether TNFalpha and Bcl-2 could modulate the activity of NFkappaB in ventricular myocytes. Stimulation of myocytes with TNFalpha resulted in a 2.1-fold increase (p < 0.001) in NFkappaB-dependent gene transcription and nuclear DNA binding. Similarly, a 1.9-fold increase (p < 0.0002) in NFkappaB-dependent gene transcription was observed in myocytes expressing Bcl-2. Nuclear DNA binding activity of NFkappaB was significantly increased in myocytes expressing Bcl-2, with a concomitant reduction in IkappaBalpha protein level. The Bcl-2-mediated loss of IkappaBalpha could be prevented by the proteasome inhibitor lactacystin, consistent with the notion that the targeted degradation of IkappaBalpha consequent to overexpression of Bcl-2 utilizes the ubiquitin-proteasome pathway. This was further tested in human 293 cells in which the N-terminal region of IkappaBalpha was identified to be an important regulatory site for Bcl-2. Deletion of this region or a serine to alanine substitution mutant at amino acids 32 and 36, which are defective for both phosphorylation and degradation, were more resistant than wild type IkappaBalpha to the inhibitory effects of Bcl-2. To our knowledge, this provides the first evidence for the regulation of IkappaBalpha by Bcl-2 and suggests a link between Bcl-2 and the NFkappaB signaling pathway in the suppression of apoptosis.


Subject(s)
Apoptosis/physiology , DNA-Binding Proteins/metabolism , I-kappa B Proteins , Myocardium/metabolism , NF-kappa B/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Acetylcysteine/analogs & derivatives , Acetylcysteine/pharmacology , Animals , Animals, Newborn , Apoptosis/drug effects , Cell Line , Cells, Cultured , Cycloheximide/pharmacology , Cysteine Endopeptidases/metabolism , Cysteine Proteinase Inhibitors/pharmacology , DNA/metabolism , Heart Ventricles , Humans , Multienzyme Complexes/metabolism , Myocardium/cytology , NF-KappaB Inhibitor alpha , NF-kappa B/antagonists & inhibitors , Proteasome Endopeptidase Complex , Rats , Rats, Sprague-Dawley , Transcription, Genetic/drug effects , Transcription, Genetic/physiology , Transfection , Tumor Necrosis Factor-alpha/pharmacology , Tumor Necrosis Factor-alpha/physiology
17.
Electrophoresis ; 19(8-9): 1351-5, 1998 Jun.
Article in English | MEDLINE | ID: mdl-9694280

ABSTRACT

The application of nonradioactive RNA probes for Northern blotting offers the advantage of a rapid turn-around time for results without the loss of sensitivity for target mRNA detection. However, a problem that has impeded the widespread use of nonradioactive RNA probes for use in Northern blotting is the difficulty in stripping these probes from nylon membranes after hybridization. In this report we describe two protocols for stripping digoxigenin (Dig)-labeled RNA probes from nylon membranes. One protocol utilizes a phosphate-buffered formamide stripping solution to remove nonchemically modified (regular) RNA probes while the other method utilizes strippable probes that were produced with a chemically modified nucleotide (CTP) and removed by a specific stripping solution. This latter method was developed by Ambion Inc. and is called Strip-EZ. We also describe a protocol for the detection of two separate rat mRNAs using both biotin and digoxigenin-labeled RNA probes that does not require stripping the membrane after hybridization. Finally, we describe the use of another new labeling technology, called Chem-Link, that quickly and conveniently labels RNA for use in Northern blotting.


Subject(s)
Biotin , Blotting, Northern/methods , Digoxigenin , Indicators and Reagents , RNA Probes , Animals , Male , Rats , Rats, Sprague-Dawley
18.
Brain Res Brain Res Protoc ; 2(4): 339-51, 1998 Jun.
Article in English | MEDLINE | ID: mdl-9630715

ABSTRACT

Non-radioactive in situ hybridization is a sensitive method for determining the site of production for secretory molecules such as cytokines. We report here on the central and peripheral induction of proinflammatory cytokines by endotoxin, and outline procedures for the generation and application of rat-specific digoxigenin (Dig)-labelled RNA probes for the localization of mRNA by in situ hybridization. Rats were injected either intravenously (i.v.) or intracerebroventricularly (i.c.v.) with vehicle or lipopolysaccharide (LPS) and sacrificed at various time intervals post-injection. Rats were then perfused with 4% paraformaldehyde and the spleens and brains were removed and cryoprotected in 30% sucrose. Dig-labelled, rat-specific, antisense and sense RNA probes were generated by in vitro transcription from PCR-derived templates. Positive staining with all the antisense probes was cytoplasmic, whereas the sense probes showed no staining. Numerous tumor necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1beta) mRNA positive cells were observed in the marginal zone and in the red pulp of the spleen after iv LPS injections, whereas sections from saline-treated animals showed minimal cytokine mRNA expression. Cells positive for TNF-alpha and IL-1beta mRNA were detectable in the brain after i.c.v. injections of LPS, but not after icv injection of vehicle. An antisense probe for c-fos was utilized in these studies as a positive control for our procedure due to its anatomically specific expression in the rat brain after LPS. In conclusion we have demonstrated that in situ hybridization with Dig-labelled RNA probes is an efficient, sensitive and reliable tool to localize cytokine mRNA production in rat tissue.


Subject(s)
Brain Chemistry/physiology , Cytokines/genetics , Digoxigenin/chemistry , RNA Probes , RNA, Messenger/analysis , Spleen/chemistry , Actins/genetics , Animals , In Situ Hybridization , Injections, Intraventricular , Interleukin-1/genetics , Lipopolysaccharides/pharmacology , Male , Rats , Rats, Sprague-Dawley , Tumor Necrosis Factor-alpha/genetics
19.
J Exp Med ; 187(4): 487-96, 1998 Feb 16.
Article in English | MEDLINE | ID: mdl-9463399

ABSTRACT

We report that chlamydiae, which are obligate intracellular bacterial pathogens, possess a novel antiapoptotic mechanism. Chlamydia-infected host cells are profoundly resistant to apoptosis induced by a wide spectrum of proapoptotic stimuli including the kinase inhibitor staurosporine, the DNA-damaging agent etoposide, and several immunological apoptosis-inducing molecules such as tumor necrosis factor-alpha, Fas antibody, and granzyme B/perforin. The antiapoptotic activity was dependent on chlamydial but not host protein synthesis. These observations suggest that chlamydia may encode factors that interrupt many different host cell apoptotic pathways. We found that activation of the downstream caspase 3 and cleavage of poly (ADP-ribose) polymerase were inhibited in chlamydia-infected cells. Mitochondrial cytochrome c release into the cytosol induced by proapoptotic factors was also prevented by chlamydial infection. These observations suggest that chlamydial proteins may interrupt diverse apoptotic pathways by blocking mitochondrial cytochrome c release, a central step proposed to convert the upstream private pathways into an effector apoptotic pathway for amplification of downstream caspases. Thus, we have identified a chlamydial antiapoptosis mechanism(s) that will help define chlamydial pathogenesis and may also provide information about the central mechanisms regulating host cell apoptosis.


Subject(s)
Apoptosis , Caspases , Chlamydia Infections/pathology , Cysteine Endopeptidases/metabolism , Cytochrome c Group/metabolism , Mitochondria/enzymology , Animals , Caspase 3 , Chlamydia Infections/enzymology , Enzyme Activation , HeLa Cells , Humans , Hydrolysis , Mice , Microscopy, Confocal , Microscopy, Fluorescence , Poly(ADP-ribose) Polymerases/metabolism
20.
Am J Physiol ; 273(2 Pt 2): R609-14, 1997 Aug.
Article in English | MEDLINE | ID: mdl-9277545

ABSTRACT

We tested whether prostaglandin synthesis mediates the lipopolysaccharide (LPS)-induced increase in splenic sympathetic nerve activity. Sprague-Dawley rats were pretreated with intravenous or intracerebroventricular injections of indomethacin, and splenic nerve activity was recorded after intravenous injections of LPS. In vehicle-pretreated rats, 100 micrograms LPS induced a 62.8 +/- 5.6% increase in splenic nerve activity beginning 22.7 +/- 2.7 min postinjection. All vehicle-pretreated animals responded to high (100 micrograms, 5 of 5 animals) and low (10 micrograms, 8 of 8 animals) doses of LPS. Both intravenous (15 mg/kg) and intracerebroventricular (50 micrograms) pretreatments with indomethacin delayed (F1.19 = 30.66, P < 0.001) the increase in nerve activity after 100 micrograms LPS. When given intravenously, 50 micrograms indomethacin (the intracerebroventricular dose) did not delay the response to intravenous LPS, indicating that the effects of intracerebroventricular indomethacin pretreatment were restricted to the central nervous system. Importantly, intracerebroventricular indomethacin reduced (2 of 7 animals) or completely blocked (5 of 7 animals) the splenic nerve response to the low dose of LPS (10 micrograms, iv). The indomethacin effects could not be accounted for by central release of vasopressin because intracerebroventricular injection of indomethacin did not alter baseline nerve activity or blood pressure, whereas intracerebroventricular injection of vasopressin rapidly increased both measures. Additionally, central injection of LPS did not elevate splenic nerve activity, whereas intracerebroventricular injection of prostaglandin E2 induced a rapid (2.2 +/- 2.7 min) increase in splenic nerve activity. These data indicate that central prostaglandin synthesis is an intermediate step whereby systemic LPS elicits an increase in sympathetic outflow to an immune organ.


Subject(s)
Brain/metabolism , Endotoxins/pharmacology , Prostaglandins/biosynthesis , Spleen/innervation , Sympathetic Nervous System/drug effects , Sympathetic Nervous System/physiology , Animals , Electrophysiology , Indomethacin/pharmacology , Injections, Intravenous , Injections, Intraventricular , Lipopolysaccharides/pharmacology , Male , Rats , Rats, Sprague-Dawley
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